Herpes Simplex Viruses types 1 and 2 (HSV-1 and HSV-2) are two members of the family Herpesviridae, which is defined by the architecture of the virion. B. Roizman, “Herpesviridae: A Brief Introduction” in Fields Virology, 2d ed., Vol. 2, pp. 1787-1793 (B. N. Fields and D. M. Knipe, eds. 1990). HSV-1 and HSV-2 are both members of the subfamily called the Alphaherpesvirinae, both are grouped in the E class of genome structure of the viruses comprising the family Herpesviridae, and both have a genome size of 152 kilobase pairs. HSV-1 and HSV-2 are closely related and have strong similarities in genome structure and at the nucleotide level. See McGeoch et al., J. Gen. Virol. 72:3057-3075 (1991). HSV-1 and HSV-2 are distinguishable in several aspects, including their G+C content of 67 mole % and 69 mole %, respectively. Also, the sequences in the HSV-1 and HSV-2 RL regions are more divergent than in the UL and US regions. McGeoch et al., J. Gen. Virol. 72:3057-3075 (1991). HSV-1 and HSV-2 also differ in restriction enzyme cleavage sites and in the sizes of viral proteins. Roizman & Sears, “Herpes Simplex Viruses and Their Replication” in Fields Virology, 2d ed., Vol. 2, pp. 1795-1817 (B. N. Fields and D. M. Knipe, eds. 1990).
HSV-1 infections are extremely common and affect from 70-80 percent of the total population in the United States. HSV-1 is transmitted via oral secretions, respiratory droplets or direct oral contact, and results in lesions or blisters on the mouth and lips. HSV-2 is transmitted venereally and causes ulcers and lesions on the genitals and surrounding areas, which can result in urinary retention, neuralgia and meningoencephalitis. Both HSV-1 and HSV-2 can cause either oropharyngeal or genital lesions that are indistinguishable.
There are many HSV-2 viral strains currently known, including HSV-2 strains G, HG52, and 333. The nucleotide sequences encompassing UL55, UL56 and a proposed ICP34.5 of HSV-2 strain HG52 have been sequenced. McGeoch et al., J. Gen. Virol. (1991). A restriction map for the HSV-2 strain HG52 has also been published in Chartrand, et al., J. Gen. Virol. 52:121-133 (1981), which is incorporated herein by reference. The HSV-2 strains G and 333 have the same BamHI, BspEI, EcoRI and Hind III restriction maps in the locale of the tk gene. The HSV-2 HG52 and G strains also have the same NcoI and BsgI restriction maps in the locale of the UL55 and UL56 genes. In the locale of the proposed HSV-2 ICP34.5 gene, HSV-2 strains HG52 and G have very similar EcoO109I and SphI restriction sites. The HSV-2 virus includes all viral strains that have been classified as HSV-2 by the Herpesvirus Study Group of the International Committee on the Taxonomy of Viruses (ICTV). See e.g., Roizman B et al., Herpesviridae, Definition, provisional nomenclature and taxonomy in Intervirology 16:201-217 (1981).
HSV-2, like other herpesviruses, has the ability to establish both a primary and a latent infection in its host. During the primary infection, HSV-2 infects the skin and epithelial cells and then spreads to the ganglia of the peripheral nervous system. After the lesions from the primary infection have healed, the HSV-2 viral DNA can remain dormant in the ganglia. This dormant or inert state is referred to as a state of latency. Periodically, the HSV-2 can become reactivated and cause lesions around the initial site of infection. During the recurrent disease episodes, the infectious HSV-2 virus particles are shed from the lesions. From a clinical perspective, this recurrence of HSV-2 infection is particularly problematic because it can occur up to ten times per year, can cause severe physical and psychological discomfort and creates the risk of infecting the patient's sexual partners. In certain individuals, recurrent infections may be asymptomatic, which can lead to inadvertent HSV-2 infection of others.
The number of individuals infected with HSV-2 in the United States is estimated to range from 40 to 60 million, and from 0.5 to 1 million new cases of genital herpes are diagnosed annually in the United States. See R. Whitley and J. Gnann, “The Epidemiology and Clinical Manifestations of Herpes Simplex Virus Infections” in The Human Herpesviruses, pp. 69-105 (Roizman, B., R. J. Whitley and C. Lopez eds., 1993). HSV-2 infection worldwide continues to increase.
Two groups that suffer the most severe forms of herpetic diseases caused by HSV-2 are infants or immunocompromised individuals. HSV-2 infection of neonates can result in encephalitis, skin lesions, keratoconjunctivitis, widely disseminated infections, microcephaly or hydranencephaly. Neonatal HSV-2 infection is almost always symptomatic and frequently lethal. Herpes simplex virus infections of the genital tract are also of special concern because of the possibility that genital ulceration may facilitate the transmission of human immunodeficiency virus (HIV) Holmberg et al., JAMA, 19,259:1048-50 (1988).
Currently, the major therapeutic treatment for recurrent HSV-2 infections is administration of acyclovir, which reduces the duration and severity of primary infection as well as the frequency of recurrence, but does not prevent asymptomatic viral shedding or the establishment of latency. The high incidence of HSV-2 infection, recurrent disease episodes, and asymptomatic transmission suggest that the best treatment will be a prophylactic treatment capable of preventing or ameliorating HSV-2-related diseases or conditions.
A number of different approaches to the development of HSV vaccines have been attempted, including live, attenuated HSV viruses, live virus vectors, killed virus vaccines and subunit protein vaccines. See R. L. Burke, “Current Status of HSV Vaccine Development” in The Human Herpesviruses, pp. 367-379 (B. Roizman, R. J. Whitley, and C. Lopez eds., 1993). A live virus vaccine is distinguishable from a killed virus vaccine in that the live virus is able to replicate, whereas the killed virus preparations are inactivated with, e.g., phenol, formaldehyde, heat or ultraviolet light, and are unable to replicate. Thus, the term “live” when used to describe a virus means that it is capable of replication. An attenuated virus is one that does not cause physical signs of disease and reduces person-to-person dissemination. An attenuated virus may still be capable of establishing latency. The advantage of a live, attenuated HSV-2 virus vaccine is that the live, attenuated HSV-2 virus can present a range of viral antigens to the host and stimulate both cell-mediated and humoral immune responses, which are both important in protection against HSV-2-related diseases and conditions. See S. C. Inglis, “Challenges and progress in developing herpesvirus vaccines,” Tibtech vol. 13, pp. 135-142 (April 1995). Attempts at producing an effective HSV-2 subunit vaccine have been unsuccessful to date.
Two of the most comprehensively developed live, attenuated HSV vaccines are recombinant derivatives of HSV-1 strain F, called R7017 and R7020. Meignier et al., J. Infect. Dis., 158:602-614 (1990). R7020 consists of the HSV-1 strain F genome having selected deletions and insertions. Results of human vaccine trials with R7020 indicate that while it is extremely safe, it is over attenuated for purposes of eliciting immunological protection against HSV-2 in humans. Thus, there remains a need in the art for a live, attenuated viral composition for the prophylactic treatment of HSV-2.
Little is known about the functions of the UL55 and UL56 gene products, except that they appear to be nonessential genes and do not share any sequence homology suggesting functional similarity. Nash & Spivack, Virology 204:794-798 (1994). The sequence of the UL55 and UL56 genes of HSV-2 strain HG52 has been described by McGeoch, J. Gen. Virol. 72:3057-3075 (1991) and is incorporated herein by reference. The UL56 gene of HSV-1 strain F has been cloned and expressed to produce recombinant polypeptides that are immunoreactive with antibodies in human HSV-1 IgM-positive sera. See, Kehm et al., Virus Research 33:55-56 (1994).
The HSV genome contains two copies of the γ134.5 nucleotide sequence that encodes the Infected Cell Protein 34.5 (“ICP34.5”). There is one γ134.5 nucleotide sequence in each of the inverted repeats flanking the long unique sequence of HSV-1. Chou et al., Science 250:1262-1266 (1990). A proposed sequence of ICP34.5 in HSV-2 strain HG52 has been disclosed. McGeogh, J. of Gen. Vir. 72:3057-3075 (1991), which is herein incorporated by reference. The ICP34.5 nucleotide sequence is predicted to encode a protein of 261 amino acids. In contrast, the HSV-1 ICP34.5 sequence is predicted to encode a protein of 263 amino acids. Although the γ134.5 open reading frame is conserved among the HSVs, the γ134.5 open reading frame is not highly conserved among other members of the herpesvirus family.
Protein Kinase R (PKR) is an important component of host responses to infection (e.g., viral infection). The enzyme is inactive in uninfected unstressed cells, but can be induced by double-stranded RNA and interferon. Upon activation, PKR phosphorylates key proteins, among which is the α subunit of eukaryotic elongation factor 2 (eIF-2α). Activation of PKR is a common mechanism by which eukaryotic cells respond to the presence of and gene expression by infectious agents. The PKR cascade curtails viral replication and thereby spares the organism in the interim between infection and immune response. See, e.g., Cassady et al. (J. Virol, 76:942-949 (2002).
It has previously been reported that, in HSV-1, the γ134.5 gene product inhibits the phosphorylation of eIF-2α, thereby enabling uninterrupted viral protein synthesis. It has also been reported that the Us11 gene product of HSV-1 can block the shutoff of protein synthesis by inhibiting phosphorylation of eIF-2α. See, Cassady et al. (J. of Vir., 72:8620-8626 (1998)), which is incorporated by reference herein. While the mechanism by which the Us11 gene product acts on eIF-2α phosphorylation is unknown, there is evidence that Us11 can compensate for certain γ134.5 knockouts in HSV-1. See, Id.
Little is known about the UL 43.5 gene product other than it appears to be in accessory protein associated with structures involved in HSV-1 capsid assembly. See, e.g., Ward et al., J. of Vir., 70:2684-2690 (1996)).